Bifidobacterium longum is a Gram-positive anaerobic bacterium and which has a genome with a high GC content (see e.g., Non-Patent Document 1). This Bifidobacterium longum is nonpathogenic and constitutes the most part of normal microflora in the large intestines of humans and other animals (see e.g., Non-Patent Document 2). This microorganism is said to have properties of promoting host's health such as enhancement of immunoreaction (see e.g., Non-Patent Document 3), inhibitory effect on the onset of cancer (see e.g., Non-Patent Document 4), protection of hosts against viral infection (see e.g., Non-Patent Documents 5 and 6), and possibility of producing antibacterial substance (see e.g., Non-Patent Document 7). Some microorganisms of the genus Bifidobacterium are widely used in the world in the preparation of fermented dairy products.
Furthermore, plasmids of Bifidobacterium are expected to be applied to probiotics vectors and oral vaccine vectors against infectious disease. Recent reports have revealed that Bifidobacterium longum is accumulated in hypoxic solid tumor after systemic administration (see e.g., Non-Patent Documents 8 and 9), and that a recombinant plasmid pBLES100-S-eCD that bears Escherichia coli codA fused with a Bifidobacterium longum hup promoter expresses cytosine deaminase in microorganisms (see e.g., Patent Document 1 and Non-Patent Documents 10 and 11). This confirmed the theory that recombinant Bifidobacterium longum is effective for enzyme-prodrug therapy. However, while these plasmids are getting attention in the fields of foods, pharmaceutical drugs, and industry, their genetic properties are little known due to the lack of an efficient replicable gene transfer system.
pBLES100, which was used in the construction of the recombinant plasmid pBLES100-S-eCD, is a shuttle vector constructed from the plasmid pTB6 of a Bifidobacterium longum BK51 and the plasmid pBR322 of Escherichia coli (see e.g., Non-Patent Document 12). This shuttle vector pBLES100 transformed Bifidobacterium longum at an efficiency of 2.2×104 transformants/μg DNA and was stable in the cells in terms of structure and segregation of phenotypes (see e.g., Non-Patent Document 13). However, as the plasmid having unmodified DNA can be cleaved with a restriction enzyme in the microorganism during transfection, cloning of a foreign gene requires higher transformation efficiency.
Patent Document 1: Japanese Laid-Open Patent Application No. 2002-97144
Non-Patent Document 1: Scardovi, Bergey's Manual of Systematic Bacteriology vol 2, eds. Sneath et al., pp. 1418-1434 (1986)
Non-Patent Document 2: Mitsuoka, Elsevier Applied Science, pp 69-114 (1992)
Non-Patent Document 3: Yasui et al., J. Dairy Sci., 74, 1187-1195 (1991)
Non-Patent Document 4: Reddy et al., Cancer Res., 53, 3914-3918 (1993)
Non-Patent Document 5: Duffy et al., Pediatr. Res., 35, 690-695 (1994)
Non-Patent Document 6: Saaverdra et al., Lancet., 344, 1046-1049 (1994)
Non-Patent Document 7: Ibrahim et al., J. Food Prot., 56, 713-715 (1993)
Non-Patent Document 8: Yazawa et al., Cancer Gene Ther., 7, 269-274 (2000)
Non-Patent Document 9: Yazawa et al., Breast Cancer Res. Treat., 66, 165-170 (2001)
Non-Patent Document 10: Nakamura et al., Biosci. Biotechnol. Biochem., 66, 2362-2366 (2002)
Non-Patent Document 11: Fujimori et al., Curr. Opin. Drug Discov. Devel., 5, 200-203 (2002)
Non-Patent Document 12: Matsumura et al., Biosci. Biotechnol. Biochem., 61, 1211-1212 (1997)
Non-Patent Document 13: Matsumura et al., Biosci. Biotechnol. Biochem., 61, 1211-1212 (1997)